Large bowel cancer, or colorectal cancer (CRC) is the third most common cause of cancer worldwide and the fourth biggest cause of cancer mortality. Twin studies have shown that the heritable contribution is ~35%, with ~5% of cases due to rare, high-penetrance mutations. In the last decade, the use of genome-wide association studies on large, well-characterised case-control cohorts of CRC has facilitated the identification of over 25 common genetic variants that carry with them an increased predisposition to colorectal cancer, invoking the common-disease common variant paradigm. As almost all of these variants lie within non-coding regions, the underlying causal mechanism is to-date poorly understood for the majority of these loci, and it is thought that they mediate risk by influencing gene expression levels. To test this hypothesis, an agnostic approach that utilises expression quantitative trait loci (eQTL) analysis was first carried on 115 normal colorectal mucosa samples and 59 peripheral blood mononuclear cells (PBMC). As these heritable variation on gene expression are likely to be subtle, there is a strong emphasis on the technical methodology to minimise experimentally-induced non-biological variations, including the extraction of high-quality RNA from primary tissue, the selection and validation of reference genes for normalisation of gene expression quantification, as well as internal validation of the samples and data processing. Thereafter, the association between the 25 CRC risk variants and the expression of their cis-genes were examined systematically, demonstrating that ten of these variants are also tissue-specific eQTLs. This intermediate phenotype strongly suggests that they confer risk, at least in part, by modifying regulatory mechanisms. One of the best eQTL associations (Xp22.2) is investigated in further detail to reveal a novel indel polymorphism (Indel24) at the distal promoter region of target gene SHROOM2 that influenced both transcript abundance and CRC risk more than the original tagging SNP. Functional verification with gene reporter assays indicated that Indel24 displays differential allelic control over transcriptional activity. Further in silico analysis and mutations to the reporter gene constructs provided evidence that Indel24 modulates transcription by modifying the spacing between CCAAT motifs and the consequent binding affinity of NF-Y transcription factor. siRNA depletion of NF-Y was associated with a reduction in transcriptional activity of the Indel24 gene construct as well as endogenous SHROOM2, which is strongly supportive of the interaction between Indel24 and NF-Y in the transcriptional activation of SHROOM2. Preliminary evidence is suggestive of SHROOM2 being expressed at the top of the intestinal epithelial crypt and playing a role in cell cycle regulation. Hypothesis-driven approaches can also be of utility in demonstrating functionality of CRC risk variants, complementing the hypothesis-free approach of eQTL analysis. Guided by a recently discovered gene-environment interaction between the 16q22.1 risk variant and circulating vitamin D levels, the influence of the rs9929218 SNP on CDH1 gene expression was examined, in relation to the expression of putative regulatory genes derived from in silico analysis and studies of other target genes. Although there was no direct association between rs9929218 and CDH1 expression, there were multiple two-way interactions that were together suggestive of rs9929218 influencing the VDR/FOXO4 regulation of CDH1. This provides functional support for the mechanism underlying the epidemiological observation of the gene-environment interaction between 16q22.1 and vitamin D, and demonstrates a candidate-based approach in deciphering the link between genetic locus and CRC susceptibility. In summary, the research presented in this thesis has validated the experimental rationale of utilising expression studies of normal colorectal mucosa to hone in on the molecular mechanisms and susceptibility genes underlying the association between common genetic variation and CRC risk.

Colorectal cancer (CRC) is the third most common cancer globally with around 1.3 million cases diagnosed annually. In cases of inherited CRC where none of the rare, high-risk mutations associated with familial syndromes are observed it has been theorised that the heritable risk is due to common, low-risk variants in the genome. Identifying these variants of modest or small effect size has become possible due to the use of large genome-wide association studies (GWAS). Through a meta-analysis of five previous GWAS, Dunlop et al. identified three novel susceptibility loci at 6p21, 11q13.4 and Xp22.2. The aims of this study were to further characterise the Xp22.2 risk locus and investigate the function of the putative risk gene SHROOM2. The variant originally identified in the Dunlop et al. study as showing an association with CRC risk rs5934683 is located between SHROOM2 and GPR143. Genome–wide expression analysis has shown that the variant is an eQTL (expression quantitative locus), affecting expression of SHROOM2, but not GPR143. An important caveat to this analysis was that the commercial array used to measure gene expression does not detect all predicted GPR143 transcripts. Hence it was important to understand whether GPR143 might be involved at the locus and whether there was altered expression in normal colonic mucosa. I have analysed the expression of GPR143 and shown that it is poorly expressed in normal mucosa and that expression of the alternative transcripts is rare. This provided further evidence for the gene of interest at Xp22.2 being SHROOM2 and thus became the focus for further investigation. In order to understand SHROOM2 function a knockout mouse was generated allowing studies of gene function beyond the previously used in vitro systems. Embryonic stem cells containing a Shroom2 knockout first allele were obtained from the International Knockout Mouse Consortium and a novel mouse line established. This mouse line was found to be an incomplete knockout and a Cre recombination strategy was employed to remove the critical exon and create a true null allele for Shroom2. This model was validated as being a true knockout of Shroom2 at both the RNA and protein level and the model subjected to initial phenotyping focusing on tissues where the gene has previously been identified as expressed. To investigate the role of Shroom2 as a CRC susceptibility gene preliminary data has been gathered from crosses to the ApcMin/+ CRC model, and analysis of the intestines of the Shroom2KO line has been undertaken. Two spontaneously occurring anorectal adenomas have been identified in Shroom2 null mice, and an additional mid-colonic polyp phenotype identified when crossed onto the ApcMin/+ background. Additionally, embryonic fibroblasts have been used in growth and wound healing assays to determine what effect total loss of Shroom2 has at a cellular level. Proteomics analysis to identify significantly altered pathways associated with Shroom2 loss has also been carried out and has highlighted a number of interesting targets for further investigation. In summary, a novel Shroom2 knockout mouse model has been developed to investigate the CRC susceptibility locus identified at Xp22.2. Preliminary data from this mouse model appears to confirm SHROOM2 as having a role in tumour development in the large intestine.

Overwhelming evidence indicates that aspirin (ASA) and related non-steroidal anti-inflammatory drugs (NSAIDs) have anti-tumour activity against colorectal cancer (CRC). Although the underlying mechanisms have yet to be fully elucidated, the host laboratory have shown that nucleolar sequestration of the NF-κB component RelA is critical. In the course of these studies, it was noted that alongside effects on the NF- κB pathway, ASA has a profound effect on nucleoli, including a dramatic increase in nucleolar size. These data were particularly interesting as, in addition to its role in ribosome biogenesis, the nucleolus is known to act as a stress sensor and play a key role in the regulation of cell growth and apoptosis. Indeed, this organelle has been identified as a potential target for anti-tumour agents. However, how stress causes changes to nucleolar function, and how these are translated into changes in cell phenotype, remain unclear. Therefore, the aim of my thesis was to fully characterise ASA effects on nucleoli and to determine whether these effects contribute to the anti-tumour activity of this agent. I found that ASA induced an atypical form of nucleolar stress that was associated with enlargement of the organelle, relocalisation of nucleolar markers to the periphery, depletion of the critical component of the Pol I transcription factor complex, TIF-IA, and inhibition of rRNA transcription. These effects were independent of the p38 and JNK2 MAP kinase pathways. However, they were mimicked by inhibition of CDK4, which had previously been shown to lie upstream of ASA effects on the NF-κB pathway. These data describe a novel mechanism by which ASA, and CDK4 inhibition, may inhibit the growth of colon cancer cells. In addition to this candidate approach, I used Stable Isotope Labelling by Amino acids in Cell culture (SILAC) based quantitative proteomics to obtain a global overview of ASA effects on nucleoli of colon cancer cells. Firstly, a protocol was successfully developed to isolate pure nucleoli from SW480 CRC cell lines. This protocol was then applied to SILAC labelled cells treated with ASA for three time-points (0, 6, 10 h). In collaboration with R.T Hay and M. Tatham (University of Dundee), proteomic analysis was then carried out by tandem-mass spectrometry. These data confirmed that ASA has a significant effect on the nucleolar proteome. They also revealed that ASA induces a distinct type of nucleolar stress that is associated with the accumulation of chaperones, translational regulators and members of the ubiquitin-proteasome system (UPS) in this organelle. These data were reminiscent of studies previously published on the effect of proteasome inhibition on nucleoli. I therefore used SILAC-based proteomics to compare ASA effects on nucleoli to those induced by the proteasome inhibitor, MG132. I found that similar sub-groups of proteins accumulate in nucleoli in response to both agents and that ASA induced proteotoxic stress in a similar manner to MG132. Fluorescence correlation spectroscopy in collaboration with R. Duncan and K. Martin (Heriot-Watt University) demonstrated the relative reduction in mobility of nucleolar DsRed-RelA, indicating that, similar to MG132, ASA induces formation of nucleolar aggresomes. Mechanistic studies suggested that blocking ASA-mediated proteotoxic stress blocked the apoptotic effects of the agent. Taken together, these data define a distinct type of nucleolar stress that may be involved in the cells response to proteotoxic stress and be required for the anti-tumour activity of ASA.

Aim: To assess SELDI-TOF MS technology as a tool for biomarker discovery in the stool and serum of colorectal cancer patients. Materials and Methods: 1.Initially a technique of analysis was developed and optimised using tumour samples and matched normal mucosa, obtained from the Tayside Tissue Bank. These samples were then analysed using SELDI on a PBS II Protein Chip Mass Spectrometer to identify abundant proteins. 2. A technique of stool preparation and subsequent SELDI analysis was developed and then optimised (CM10 chip at pH4) to allow comparison of faecal samples from cancer and controls. Faecal samples were then collected from cancer patients and controls and analysed. In addition, FOB testing was carried out on all stool samples from cancer and controls and subgroup analysis of spectras was performed controlling for FOB status. 3. A test set of cancer and normal serum samples was used to optimise the method of analysis using 4 different chip surfaces at differing pH. Serum samples were collected from cancer patients and normal controls and were analysed on the H50 chip. Serum was then depleted of major proteins in an attempt to improve the detection of peaks. The mass spectra from each sample type were compared to identify any common protein peaks. Results: 1. Tumour analysis methods were optimised using an initial 4 samples of tumour and normal mucosa. Analysis of 8 further paired samples showed protein peaks at 2826, 3374, 3444, 3489 and 10854 Da which were abundant in tumour and reduced in the normal mucosa. 2. In serum analysis the initial experiment of 10 cancer versus 10 normal revealed 4 peaks on the H50 chip (3479, 3364, 3434, 3700 Da) that had significantly higher mass to charge ratios in cancer. The experiment was repeated on the H50 chip using 92 cancers and 92 controls and 5 different peaks were identified (7901, 8124, 8566, 8799 and 17 409Da) as being significant but these had higher mass to charge ratios in the controls. After depletion of the serum samples of albumin, transferrin, haptoglobin, anti-trypsin, IgG and IgA SELDI-TOF analysis showed a greatly reduced profile that yielded no meaningful spectra. 3. Stool analysis revealed 5 protein peaks (4633, 16511, 33423, 37087 and 47026 Da) in colorectal cancer patients, which were absent in stools from controls with a sensitivity of 83% when using all 5 peaks. Degradation of the spectra was observed after prolonged storage of stool samples. Conclusions: A method of stool analysis has been developed that yielded valid peaks differentiating between cancer and normal, which warrant further research through protein identification. Serum analysis was not reproducible across experiments and depletion of major proteins failed to reveal the sub-proteome raising doubts about whether discovery-based serum proteomics can accurately detect cancer. SELDI-TOF was not able to demonstrate that any of the peaks present in the tumour analysis were present in the stool or the serum samples.

Colorectal cancer (CRC) is a primary cause of cancer-related mortality in the United States. Asian Americans have the highest CRC mortality rates. CRC screening tests can reduce CRC incidence, yet Asian Americans, specifically the subgroup of Vietnamese Americans, underuse CRC screening. The purpose of this phenomenological study was to understand why Vietnamese Americans, ages 50 to 75, underuse CRC screening. The health belief model constructs of susceptibility, severity, benefits, barriers, and self-efficacy were the framework for understanding this population's health-related behaviors. Three research questions focused on how knowledge, language, and cultural beliefs and perceptions affect Vietnamese Americans' CRC screening decisions. Interviews were conducted with 11 participants, and transcribed interview responses were input into NVivo 11 software to maintain a reliable database and to identify emerging themes. Key study findings revealed knowledge and English language gaps as well as adverse cultural perceptions of fear and doubt that influenced CRC screening choices among these 11 Vietnamese Americans. Future researchers might focus on cultural-tailored strategies to minimize these barriers for Vietnamese Americans. An understanding of this study population's perspectives offers the promise of positive social change for health services and public health administrations to develop cultural-tailored interventions that promote healthy lifestyles, prevention, early CRC detection and, consequently, reduce mortality rates and associated health care costs.

DNA methylation is increasingly recognised as a significant epigenetic event that may initiate and drive the process of neoplasia in humans. In the colon, DNA methylation of key genes is common in a subset of colorectal cancers. The extent to which DNA methylation at various genes contributes to initiation of colorectal neoplasms is less clear. This study sought to clarify the biological and clinicopathological significance of methylation of various genes in the development of sporadic and familial colorectal neoplasia. Quantitative methylation-specific PCR (qMSP) assays (capable of detecting down to a measureable proportion of 0.1% of the total input DNA) were developed to determine the presence of CpG methylation at a given gene. Methylation of MLH1-C was found in the apparently normal mucosa samples from seven of 104 (7%) of individuals with sporadic colorectal cancer (CRC) showing microsatellite instability (MSI). No methylation of MLH1-C was found in the biological samples of individuals with microsatellite stable (MSS) counterparts (n=131). MLH1-C methylation may be a field defect that predisposes to the development of sporadic colorectal neoplasia, particularly those demonstrating MSI. Methylation of three of five genes within the 3p22 region including AB002340, MLH1, ITGA9, PLCD1 and DLEC1 (regional 3p22 methylation) was found in 83% of sporadic MSI (n=86) and 12% of MSS cancers demonstrating BRAF V600E mutation (n=42). Regional 3p22 correlated strongly with CpG island methylator phenotype (CIMP), and other clinicopathological characteristics typical of CIMP. Thus, regional 3p22 methylation and CIMP may be overlapping phenomena. Regional 3p22 methylation and the BRAF V600E mutation were found in normal colonic mucosa of four individuals with sporadic MSI CRC, and these cases also had multiple synchronous serrated polyps. These molecular aberrancies may predispose some individuals to the development of metachronous serrated neoplasia. Germline epimutations of APC do not contribute towards the development of FAP, AFAP, or hyperplastic polyposis syndromes. However, APC methylation in normal colonic mucosa of these individuals may represent a field defect in the development of futher neoplasms. In conclusion, different patterns of DNA methylation in normal colonic mucosa may represent a field defect important in the development of different subtypes of colorectal neoplasia.

Colorectal cancer (CRC) is one of the most common causes of death from malignant disease. Nevertheless, no ideal screening method exists and there is a lack of prognostic and predictive factors to support clinical decisions and to aid the development of a more individualized treatment for patients with CRC. The aim of this thesis was to investigate early detection, prognostic and predictive factors of CRC. In the first paper, a novel method to collect cells for DNA quantification from the rectal mucosa was investigated. The sensitivity and specificity of this test to detect CRC or any pathology in colon and rectum were ultimately too low to be acceptable. In the second paper, the prognostic value of growth differentiation factor 15 (GDF 15) was evaluated in patients curatively operated for colorectal cancer. GDF 15 expression was demonstrated to be associated with a negative prognosis in patients with stages I-III and III disease. In the third paper, the prognostic value of BRAF, PIK3CA KRAS and MSI was evaluated in a cohort of patients with CRC stratified by disease and recurrence. The results indicated that patients with CRC stage III without recurrence have a higher frequency of BRAF mutation compared to stage III patients with recurrence. In the fourth paper, histopathological predictors of pathologic complete response (pCR) as well as the association between pre-treatment carcinoembryonic antigen (CEA) levels and pCR in non-smoking and smoking patients receiving preoperative chemo-radiotherapy for rectal cancer were evaluated. Only in non-smokers was a low CEA level significantly associated with pCR, suggesting that the predictive value of CEA for pCR in rectal cancer in smokers can be limited. In sum, this research has investigated a new method for CRC detection and further evaluated the clinical use of prognostic and predictive markers in CRC.

El càncer colorectal (CCR) és una de les neoplàsies més freqüents. La seva freqüència ha augmentat en els últims anys i constitueix la segona causa de mort per càncer en ambdós sexes. Els fàrmacs més àmpliament utilitzats en el tractament quimioteràpic dels pacients amb càncer de recte són el 5-Fluorouracil (5-FU) i el Oxaliplatí (OXA). Els estudis farmacogenètics plantegen la utilització dels coneixements derivats dels estudis genòmics per tal que els pacients es puguin sotmetre a tractaments farmacològics més eficaços i menys tòxics. Aquests estudis tenen una especial rellevància en el camp de l’oncologia, ja que els tractaments quimioradioteràpics es caracteritzen per tenir una finestra terapèutica molt estreta que fa difícil establir l’equilibri entre l’increment de la dosi (per millorar l’eficàcia) i l’aparició d’efectes adversos. Pel que fa a l’estudi de la relació de la resposta clínica i l’aparició d’efectes adversos del tractament amb 5-FU en pacients amb CCR avançat i el patró de polimorfismes/mutacions dels gens relacionats amb la via metabòlica de la síntesis de folats, les conclusions d’aquesta tesi són: 1. El nombre de llocs d’unió de factors de transcripció de la regió 5’ no traduïda del gen de la Timidilat Sintasa és un marcador farmacogenètic de l’eficàcia del tractament amb 5-FU. 2. No s’ha evidenciat una associació entre els polimorfismes del gen de la Metilentetrahidrofolat reductasa (C677T i A1298C) i la toxicitat relacionada amb l’administració de 5-FU. 3. Existeix una associació estadísticament significativa entre el genotip A1298C del gen de la Metilentetrahidrofolat reductasa i la supervivència global en les dones tractades amb 5-FU. 4. Ni els reordenaments genòmics en el gen de la Dihidropirimidina deshidrogenasa, ni la presència de la mutació IVS14 +1 G>A tenen un paper rellevant en el desenvolupament d’efectes adversos associats amb el tractament amb 5-FU. Pel que fa a l’estudi de la relació de la resposta i de la toxicitat del tractament amb derivats del platí en pacients amb CCR avançat amb el patró de polimorfismes en gens relacionats amb els mecanismes de reparació de l’ADN de les vies de reparació NER i BER, les conclusions són: - Via NER: 1. El polimorfisme C118T del gen de reparació per escissió del grup de complementació creuada 1 és un bon marcador pronòstic que avalua la resposta i la supervivència en pacients amb CCR tractats amb règims OX/5-FU. 2. El polimorfisme Lys751Gln del gen xeroderma pigmentosa grup D és un marcador pronòstic que avalua la resposta i la supervivència global en pacients tractats amb derivats del platí. - Via BER: 1. No es demostra una associació entre els tres polimorfismes del gen de reparació per raigs X del grup de complementació creuada 1 i els paràmetres clínics que defineixen la remissió clínica i la supervivència. Pel que fa a l’estudi de la resposta a la quimioteràpia amb derivats de platí en pacients amb CCR avançat en relació amb el polimorfisme de la Glutatió-S-transferasa, la conclusió és: 1. No es demostra associació significativa entre el polimorfisme Ile105Val d’aquest gen i la neurotoxicitat induïda pel tractament amb oxaliplatí. / Colorectal cancer (CRC) is one of the most frequent neoplasms. Its frequency has increased in recent years and is the second leading cause of cancer death in both sexes. The drugs most widely used in chemotherapy for patients with colorectal cancer are 5-fluorouracil (5-FU) and oxaliplatin (OXA). Pharmacogenetics studies raise the use of knowledge gained from genomic studies so that patients can be subjected to more effective and less toxic drug treatments. These studies have special relevance in the field of oncology, as chemotherapy treatments are characterized by a very narrow therapeutic window that makes it difficult to establish a balance between increasing the dose (to improve efficiency) and the occurrence of adverse effects. Regarding the study of the relationship between clinical response and the emergence of adverse effects of treatment with 5-FU in patients with advanced CRC and the pattern of polymorphisms / mutations of genes related to metabolic pathway of folate synthesis, the conclusions of this thesis are: 1. The number of binding sites of transcription factors in the 5’ UTR region of thymidylate synthase gene is a pharmacogenetics marker of the efficacy of treatment with 5-FU. 2. It has shown an association between polymorphisms methylenetetrahydrofolate reductase gene (C677T and A1298C) and toxicity associated with administration of 5-FU. 3. There is a significant association between the A1298C genotype methylenetetrahydrofolate reductase gene and overall survival in women treated with 5-FU. 4. Neither the genomic rearrangements in the dihydropyrimidine dehydrogenase gene, the presence of the mutation IVS14 +1 G> A have a role in the development of adverse effects associated with treatment with 5-FU. Regarding the study of the relationship between the response and toxicity of treatment with platinum derivatives in patients with advanced CRC with the pattern of polymorphisms in genes related to the mechanisms of DNA repair pathways: NER and BER, the conclusions are: - Via NER: 1. The C118T polymorphism of the gene for excision repair cross complementing 1 is a good prognostic marker to evaluate response and survival in patients with CRC treated with regimens OX/5-FU. 2. Polymorphism Lys751G in the Xeroderma Pigmentosum group D gene is a prognostic marker to evaluate the response and overall survival in patients treated with platinum derivatives. - Via BER: 1. Not demonstrated an association between three polymorphisms of the X-ray repair complementing group 1 gene and clinical parameters that define clinical remission and survival. Regarding the study of response to chemotherapy with platinum derivatives in patients with advanced CRC in relation to the polymorphism of glutathione-S-transferase P1 gene, the conclusion is: 1. It demonstrates a significant association between the Ile105Val polymorphism of this gene and treatment with oxaliplatin-induced neurotoxicity.

This study evaluates a new serum-based biomarker for colorectal cancer (CRC) screening and diagnosis. The biomarker (GTA-446) is a member of hydroxy -polyunsaturated ultra-long chain fatty acids and was found to be reduced in CRC patients compared to CRC-free subjects. Diagnostic test performance characteristics were used to identify the effectiveness of the test. Methods: Serum levels of GTA-446 were measured in 4924 subjects who underwent colonoscopy for any reason, pathology results and clinical data were also collected. Two sets of age-matched control subjects were used; First were the lab controls (number=383) which were serum samples collected from Saskatchewan Disease Control Laboratory along with age and gender data. Second, were the endoscopy controls (number=762) which were obtained from the colonoscopy population after being determined to be cancer-free. Cut-off values were calculated using Receiver Operating Characteristic (ROC) curve. Results: Serum GTA-446 was found to be reduced in 87% of CRC patients. Compared to lab controls, the GTA-446 biomarker has a sensitivity of 87%, specificity of 75%, positive likelihood ratio of 3.6, and negative likelihood ratio of 0.16. Using endoscopy controls to calculate test performance characteristics, the biomarker has a sensitivity of 87%, specificity of 50%, positive likelihood ratio of 1.74, and negative likelihood ratio of 0.24. Also, the level of GTA-446 was found to significantly decline with age (r=-0.20, p<0.01). Conclusion: Serum GTA-446 is a potential biomarker for minimally invasive detection of colorectal cancer that compares favorably to other serum-based biomarkers.

Abstract The homeobox gene prox1 is a transcription factor related to the Drosophila gene Prospero. It play an essential role in the development of central nervous system, lens, liver and pancreas. In addition, prox1 is a master gene controlling the early development of the lymphatic vasculature. In tumorigenesis, prox1 has been shown to function as a tumor suppressor gene in hepatocellular carcinoma and breast cancer. Conversely, prox1 is over-expressed in the majority of colorectal cancer (CRC) and it promotes dysplasia, tumor growth and malignant progression. I report the findings here and show that ectopic expression of prox1 in prox1-null DLD-1 colon cancer cells will increase cell invasion but decrease cell adhesion. In addition, prox1 may induce epithelia- mesenchymal transition (EMT) by attenuating E-cadherin expression and up-regulating other EMT markers. On the contrary, knockdown of prox1 increases E-cadherin expression in SW620 cells; reduction of prox1 increases cell adhesion but decreases invasion. In short, the transcription factor prox1 plays an oncogenic role and promotes cancer metastasis in CRC.